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Enhancing Viability of Human Embryonic Stem Cells during Cryopreservation via RGD-REP-Mediated Activation of FAK/AKT/FoxO3a Signaling Pathway

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Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Background

Cryopreservation is a crucial method for long-term storage and stable allocation of human pluripotent stem cells (hPSCs), which are increasingly being used in various applications. However, preserving hPSCs in cryogenic conditions is challenging due to reduced recovery rates.

Methods

To address this issue, the Arginine-Glycine-Aspartate (RGD) motif was incorporated into a recombinant elastin-like peptide (REP). Human embryonic stem cells (hESCs) were treated with REP containing RGD motif (RGD-REP) during suspension and cryopreservation, and the survival rate was analyzed. The underlying mechanisms were also investigated.

Results

The addition of RGD-REP to the cryopreservation solution improved cell survival and pluripotency marker expression. The improvement was confirmed to be due to the activation of the FAK-AKT cascade by RGD-REP binding to hESC surface interin protein, and consequent inhibition of FoxO3a. The inactivation of FoxO3a reduced the expression of apoptosis-related genes, such as BIM, leading to increased survival of PSCs in a suspension state.

Conclusion

RGD-REP, as a ligand for integrin protein, improves the survival and maintenance of hPSCs during cryopreservation by activating survival signals via the RGD motif. These results have potential implications for improving the efficiency of stem cell usage in both research and therapeutic applications.

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Acknowledgements

This Work in Kim lab was supported by the National Research Foundation of Korea (NRF-2022M3A9H1016308 and NRF-2022R1F1A1066611), the Korean Fund for Regenerative Medicine (2021M3E5E5096744), and the KIST Institutional Program (Project No. 2Z05790-19-037). Work in Jeon lab was supported by the National Research Foundation of Korea (NRF-2019M3A9H1103478). Illustrations were created using image sources from Biorender (www.biorender.com).

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Authors and Affiliations

  1. Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul, 02447, Republic of Korea

    Jeong Hee Kim, Jeong In Choi, Young Hyun Che & Yong Jun Kim

  2. Department of Pathology, College of Medicine, Kyung Hee University, Seoul, 02447, Republic of Korea

    Su Haeng Sung, Sun Lee, Jae-Hoon Park & Yong Jun Kim

  3. Cedars-Sinai Medical Center, Biomanufacturing Center, Los Angeles, CA, 90069, USA

    Hojae Lee

  4. Well Aging Research Center, Division of Biotechnology, DGIST, Daegu, 42988, Republic of Korea

    Yun-Il Lee & Won Bae Jeon

  5. Department of Interdisciplinary Studies, DGIST, Daegu, 42988, Republic of Korea

    Yun-Il Lee

  6. Department of New Biology, DGIST, Daegu, 42988, Republic of Korea

    Young-Sam Lee

  7. KHU-KIST Department of Converging Science and Technology, Graduate School, Kyung Hee University, Seoul, 02447, Republic of Korea

    Yong Jun Kim

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Contributions

JHK: study design, performing experiments and data analysis; JIC, YHC, SHS and HL: performing experiments and data analysis; SL and JHP: data assembly and analysis; YIL and YSL: conception, study design, and data analysis; WBJ: conception and study design; YJK: conception, study design, data analysis, writing manuscript.

Corresponding author

Correspondence to Yong Jun Kim.

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Conflict of interest

Won Bae Jeon is the founder and CEO of Excellamol Inc., and has employment and financial relationships with Excellamol Inc., including patent inventions. All other authors have no financial conflicts of interests.

Ethical statement

All experiments using hESCs were conducted in accordance with the regulation KHSIRB-20–489 permitted by Kyung Hee University IRB.

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Won Bae Jeon have Corresponding to REP material.

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Kim, J.H., Choi, J.I., Che, Y.H. et al. Enhancing Viability of Human Embryonic Stem Cells during Cryopreservation via RGD-REP-Mediated Activation of FAK/AKT/FoxO3a Signaling Pathway. Tissue Eng Regen Med 20, 1133–1143 (2023). https://doi.org/10.1007/s13770-023-00568-3

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  • DOI: https://doi.org/10.1007/s13770-023-00568-3

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